Mulder H, Dideberg F, Schachter H, Spronk B A, De Jong-Brink M, Kamerling J P, Vliegenthart J F
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Eur J Biochem. 1995 Aug 15;232(1):272-83. doi: 10.1111/j.1432-1033.1995.tb20809.x.
Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (beta 2Xyl-T), beta 1-2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N-acetylglucosaminyltransferase II (beta 2GlcNAc-T II) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = -4GlcNAc beta 1-4GlcNAc or O-(CH2)7CH3] with UDP-Xyl and connective tissue microsomes have been purified and characterized by 1H-NMR spectroscopy in conjunction with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specificity studies focused on connective tissue beta 2Xyl-T show that the minimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)7CH3. The enzyme activity can therefore be characterized as UDP-Xyl:Glc-NAc beta 1-2Man alpha 1-3Man beta-R (Xyl to Man beta) beta 1-2 xylosyltransferase. In substrate-specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xyl residue at C2 of beta Man totally abolishes the enzyme activity. Xylose-containing oligosaccharides are not acceptors for beta 2GlcNAc-T I. In combination with the substrate specificity of beta Xyl-T, this shows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc-T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective tissue is proposed.
通过使用一系列相关底物，已证明椎实螺（Lymnaea stagnalis）的结缔组织含有β1-2木糖基转移酶（β2Xyl-T）、β1-2 N-乙酰葡糖胺基转移酶I（β2GlcNAc-T I）和β1-2 N-乙酰葡糖胺基转移酶II（β2GlcNAc-T II）活性。这些酶可能参与N-连接碳水化合物链的生物合成，就像血蓝蛋白中存在的那些链一样。用UDP-木糖和结缔组织微粒体孵育GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-R [其中R = -4GlcNAcβ1-4GlcNAc或O-(CH2)7CH3] 所形成的产物已通过1H-NMR光谱结合甲基化分析进行了纯化和表征，结果为GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-R。针对结缔组织β2Xyl-T的底物特异性研究表明，GlcNAcβ1-2Manα1-3Manβ1-O-(CH2)7CH3满足了最小结构要求。因此，该酶活性可被表征为UDP-木糖：Glc-NAcβ1-2Manα1-3Manβ-R（木糖转移至Manβ）β1-2木糖基转移酶。在针对结缔组织β2GlcNAc-T I的底物特异性研究中，可以证明该酶对至少具有Manα1-3Manβ-R序列的受体具有活性，并且在β-Man的C2位引入β-木糖残基会完全消除酶活性。含木糖的寡糖不是β2GlcNAc-T I的受体。结合β-木糖基转移酶的底物特异性，这表明在蜗牛结缔组织中，β2GlcNAc-T I必须在β2Xyl-T之前起作用。结缔组织β2GlcNAc-T II活性遵循先前确立的生物合成途径。基于目前已知的各种结缔组织糖基转移酶的底物特异性以及从椎实螺血蓝蛋白中分离出的结构，提出了蜗牛结缔组织中N-糖基化的部分生物合成方案。
